Methods for making hetero-dimeric proteins have been reported. The first approach to construct and produce hetero-dimeric bispecific antibodies was the quadroma technology (Milstein C and Cuello A C, Nature, 305(5934):537-40 (1983)) which consists of a somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. Because of the random pairing of two different immunoglobulin (Ig) heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different immunogloblin species are generated of which only one is the functional bispecific antibody (Kufer P et al., Trends Biotechnol, 22(5):238-44 (2004)). The presence of mispaired by-products reduces significantly the production yield and requires sophisticated purification procedures to achieve product homogeneity. The mispairing of Ig heavy chains can be reduced by using several rational design strategies, most of which engineer the antibody heavy chains for hetero-dimerization via the design of man-made complementary hetero-dimeric interfaces between the two subunits of the CH3 domain homo-dimer. The first report of an engineered CH3 hetero-dimeric domain pair was made by Carter et al. describing a “protuberance-into-cavity” approach for generating a hetero-dimeric Fc moiety (U.S. Pat. No. 5,807,706; ‘knobs-into-holes’; Merchant A M et al., Nat Biotechnol, 16(7):677-81 (1998)). Alternative designs have been recently developed and involved either the design of a new CH3 module pair by modifying the core composition of the modules as described in WO2007/110205 or the design of complementary salt bridges between modules as described in WO2007/147901 or WO2009/089004. The disadvantage of the CH3 engineering strategies is that these techniques still result in the production of a significant amount of undesirable homo-dimers. Hence there remains a need for an engineering technique which minimizes the content of homo-dimeric species.
Regardless of the various approaches at hetero-dimerizing Ig heavy chains as described in these patent publications, the major obstacle facing the development of full bispecific antibodies (i.e., two FAB fragments, each having a unique set of variable heavy and light chain domains that creates a unique antigen binding site and one dimeric Fc region) based on any CH3 domain rational engineering or others, is the requirement of having a common light chain to both FABs in order to circumvent the mispairing of their light chains (Carter P, J Immunol Methods, 248(1-2):7-15 (2001)). Although, this can be accomplished by using antibodies with identical light chains that bind to different antigens by virtue of their distinct heavy chains, it does requires the isolation of such antibodies, which usually involves the use of display technologies, and here are no current technologies that will enable the direct use of two distinct human monoclonal antibodies with the desired specificities to be reassembled without further CDR or light chain engineering into a full bispecific antibody. Thus there is a need for generating full, correctly assembled, bispecific antibodies that are similar in their overall structure to natural antibodies, i.e., comprising two FAB fragments, each having a unique set of variable heavy and light chain domains that creates a unique antigen binding site and one dimeric Fc region.